Auswahl der wissenschaftlichen Literatur zum Thema „Sumoylome“

Abstract Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, the mechanisms that orchestrate the global and dramatic transcriptional changes leading to adipocyte differentiation remain to be fully unraveled. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing endogenous sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like PPARγ/RXR and their co-factors are associated with the transcription of adipogenic genes. Finally, using RXR as a model, we found that sumoylation may regulate adipogenic transcription by supporting the chromatin occurrence of transcription factors. Our data demonstrate that the sumoylation pathway supports the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides the scientists in the field of cellular differentiation and development with an in-depth resource of the dynamics of the SUMO-chromatin landscape, SUMO-regulated transcription and endogenous sumoylation sites during adipocyte differentiation.

Post-translational modification pathways such as SUMOylation are integral to all cellular processes and tissue homeostasis. We investigated the possible involvement of SUMOylation in the epithelial signalling in Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD). Initially in a murine model of IBD, induced by dextran–sulfate–sodium (DSS mice), we observed inflammation accompanied by a lowering of global SUMOylation of colonic epithelium. The observed SUMOylation alteration was due to a decrease in the sole SUMO E2 enzyme (Ubc9). Mass-spectrometric analysis revealed the existence of a distinct SUMOylome (SUMO-conjugated proteome) in DSS mice with alteration of key cellular regulators, including master kinase Akt1. Knocking-down of Ubc9 in epithelial cells resulted in dramatic activation of inflammatory gene expression, a phenomenon that acted via reduction in Akt1 and its SUMOylated form. Importantly, a strong decrease in Ubc9 and Akt1 was also seen in endoscopic biopsy samples ( N = 66) of human CD and UC patients. Furthermore, patients with maximum disease indices were always accompanied by severely lowered Ubc9 or SUMOylated-Akt1. Mucosal tissues with severely compromised Ubc9 function displayed higher levels of pro-inflammatory cytokines and compromised wound-healing markers. Thus, our results reveal an important and previously undescribed role for the SUMOylation pathway involving Ubc9 and Akt1 in modulation of epithelial inflammatory signalling in IBD.

ABSTRACTCell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter cells are severed. Here, we present a novel abscission mechanism. We identified chromokinesin KIF4A, which is adjacent to the midbody during cytokinesis, as being required for efficient abscission. KIF4A is regulated by post-translational modifications. We evaluated modification of KIF4A by the ubiquitin-like protein SUMO. We mapped lysine 460 in KIF4A as the SUMO acceptor site and employed CRISPR-Cas9-mediated genome editing to block SUMO conjugation of endogenous KIF4A. Failure to SUMOylate this site in KIF4A delayed cytokinesis. SUMOylation of KIF4A enhanced the affinity for the microtubule destabilizer stathmin 1 (STMN1). We here present a new level of abscission regulation through the dynamic interactions between KIF4A and STMN1 as controlled by SUMO modification of KIF4A.

The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification.

Tunneling nanotubes (TNT) are thin, membranous, tunnel-like cell-to-cell connections, but the mechanisms underlying their biogenesis or functional role remains obscure. Here, we report, Rhes, a brain-enriched GTPase/SUMO E3-like protein, induces the biogenesis of TNT-like cellular protrusions, “Rhes tunnels,” through which Rhes moves from cell to cell and transports Huntington disease (HD) protein, the poly-Q expanded mutant Huntingtin (mHTT). The formation of TNT-like Rhes tunnels requires the Rhes’s serine 33, C-terminal CAAX, and a SUMO E3-like domain. Electron microscopy analysis revealed that TNT-like Rhes tunnels appear continuous, cell–cell connections, and <200 nm in diameter. Live-cell imaging shows that Rhes tunnels establish contact with the neighboring cell and deliver Rhes-positive cargoes, which travel across the plasma membrane of the neighboring cell before entering it. The Rhes tunnels carry Rab5a/Lyso 20-positive vesicles and transport mHTT, but not normal HTT, mTOR, or wtTau proteins. SUMOylation-defective mHTT, Rhes C263S (cannot SUMOylate mHTT), or CRISPR/Cas9-mediated depletion of three isoforms of SUMO diminishes Rhes-mediated mHTT transport. Thus, Rhes promotes the biogenesis of TNT-like cellular protrusions and facilitates the cell–cell transport of mHTT involving SUMO-mediated mechanisms.

Les modifications post-traductionnelles sont essentielles à la régulation des fonctions protéiques et, par conséquent, au maintien de l’intégrité cellulaire. Ces modifications interviennent à tous les niveaux de la communication neuronale. Lors de mon doctorat, j’ai étudié l’une de ces modifications, la SUMOylation. Elle consiste en la conjugaison enzymatique covalente mais réversible, de la protéine SUMO (Small Ubiquitin-like MOdifier) sur des résidus lysine spécifiques de protéines cibles. Au cours des dix dernières années, la SUMOylation est apparue comme un régulateur important de la fonction neuronale, jouant un rôle dans le développement du cerveau, l'excitabilité neuronale, la transmission et la plasticité synaptique. Il existe par ailleurs une balance entre l’état SUMOylé et déSUMOylé des protéines cérébrales, permettant une régulation fonctionnelle à la fois dans le temps et dans l’espace. Il est important de noter que les altérations du processus d'homéostasie de la SUMOylation sont associées à une variété de troubles cérébraux. Pour mieux comprendre les fonctions synaptiques de la SUMOylation, il est donc essentiel d’identifier les protéines de la synapse modifiées par SUMO ainsi que de déterminer les mécanismes qui y régulent la balance SUMO/déSUMOylation.Cependant, les mécanismes de régulation restent à élucider et le répertoire disponible des substrats SUMO identifiés au niveau des synapses est encore extrêmement limité. Mon travail de thèse a donc été découpé en deux axes : l’étude de la régulation synaptique de la balance SUMOylation/déSUMOylation et l’identification et la caractérisation d’un SUMOylome à la synapse.La balance SUMOylation/désSUMOylation est maintenue par l'action coordonnée de la seule enzyme de conjugaison SUMO, Ubc9, et des enzymes de déSUMOylation appelées SENP. Nous avons dans unp remier temps démontré qu'une activation brève des récepteurs métabotropique du glutamate mGlu5R piège transitoirement Ubc9 aux post-synapses, ce qui entraîne une augmentation rapide de la SUMOylation synaptique globale et des changements dans l'excitabilité neuronale. Une activation prolongée des récepteurs mGlu5R entraîne une accumulation post-synaptique de SENP1 et une diminution de la SUMOylation synaptique. Étant donné que les deux processus sont contrôlés par l'activation des récepteurs mGlu5R mais sur des échelles de temps différentes, d'autres mécanismes doivent coexister pour réguler cet équilibre au niveau des synapses. En utilisant une combinaison d'imagerie en temps réel et d'approches biochimiques et pharmacologiques, nous avons montré une régulation bidirectionnelle de la déSUMOylation synaptique par les mGluRs du groupe I (mGluR1 et 5) et mis en évidence un rôle de l'activation des kinases PKC et CaMKII ainsi que la polymérisation des microtubules dans la redistribution synaptique de SENP1.En combinant fractionnements subcellulaires à partir de cerveaux de rats post-nataux (P14) et immunoprécipitations SUMO2/3 spécifiques pour isoler les protéines synaptiques SUMO2/3-ylées, nous avons identifié 803 protéines synaptiques SUMOylées. Ces protéines sont importantes pour la fonction synaptique comme par exemple Synapsin3 impliquée dans l’exocytose. Parmi les nouvelles cibles identifiées, on retrouve et SynGAP, ce qui souligne le rôle central de la SUMOylation dans la fonction synaptique. De nombreuses autres protéines identifiées sont directement impliquées dans des troubles neurologiques laissant suggérer une participation du processus de SUMOylation dans l'étiologie de ces maladies cérébrales.Ainsi, mes travaux de thèse ont permis de collecter des informations importantes sur les mécanismes de régulation séquentiels dépendants de l'activité qui conduisent à l'homéostasie de la SUMOylation des protéines au niveau de la synapse et d’établir la première cartographie des cibles SUMO2/3 synaptiques
SUMOylation is a dynamic post-translational modification that consists in the covalent but reversible enzymatic conjugation of the Small Ubiquitin-like MOdifier (SUMO) protein on specific lysine residues of target proteins. In the last 10 years, SUMOylation has emerged as an important regulator of the neuronal function, playing a role in brain development, neuronal excitability and synaptic transmission and plasticity. Importantly, alterations in the SUMOylation homeostasis process are associated with a variety of brain disorders. Nevertheless, the regulatory mechanisms remain poorly studied and the available repertoire of identified SUMO substrates at synapses is still extremely limited. The SUMOylation/deSUMOylation balance is orchestrated by the coordinated action of the sole SUMO-conjugating enzyme Ubc9 and SUMO-deconjugating enzymes called SENPs. A tight regulation of this balance is therefore critical to the brain function and its disruption is associated with several neurological disorders. We previously demonstrated that a short activation of mGlu5R transiently traps Ubc9 at the post-synapse, resulting in a rapid increase in the overall synaptic SUMOylation and changes in neuronal excitability. A sustained activation of mGlu5R leads to post-synaptic accumulation of SENP1 and a decrease in synaptic SUMOylation to basal levels. Given that both processes are controlled by the activation of mGlu5R but on a different timescale, other mechanisms must coexist to regulate their spatiotemporal balance at synapses. Using a combination of advanced live-cell imaging, biochemical and pharmacological approaches, we revealed a bidirectional regulation of synaptic deSUMOylation targeting by the group I mGluRs and highlight how PKC and CaMKII activation as well as the polymerization of microtubules drive the neuronal and synaptic redistribution of SENP1. The poor identification of endogenous synaptic SUMOylation substrates is mainly due to the low levels of SUMOylated proteins at synapses but also to the developmental ages investigated. To overcome these difficulties, we combined subcellular fractionation approaches on post-natal (P14) rat brains with specific SUMO2/3 immunoprecipitation to isolate synaptic SUMO2/3-ylated proteins. Then using Orbitrap mass spectrometry (MS), we identified around 800 synaptic SUMO2/3-ylated proteins including the previously reported SUMO targets Ubc9, CASK and Synapsin1. We identified many novel synaptic SUMO targets including PSD95 and SynGAP, further highlighting the central role of SUMOylation in the synaptic function. Interestingly, many of the proteins identified are directly linked to neurological disorders suggesting that the SUMOylation process per se could participate in the aetiology of these brain diseases. Altogether, my PhD work provides additional insights into the sequential activity-dependent regulatory mechanisms driving the homeostasis of protein SUMOylation at the mammalian synapse and establishes the first detailed cartography of synaptic SUMO2/3 substrates

L'immunothérapie est récemment apparue comme une approche anti-cancéreuse prometteuse, mais ne profitant qu'à un nombre limité de patients. Étant donné que la perte de la SUMOylation dans les cellules immunitaires induit une réponse massive d'interféron de type I (IFN-I) et que l’IFN-I agit en synergie avec la thérapie par blocage des points de contrôle, nous avons étudié l'effet de la manipulation de la voie SUMO sur les réponses immunitaires anti-tumorales. Mes résultats ont révélé que des souris hypoSUMOylées présentent un délai significatif de croissance tumorale sous anti-PD-1 par rapport aux WT et que cette réponse dépend en partie de la signalisation IFN-I et implique à la fois les populations myéloïdes et lymphocytaires. De plus, nous avons défini le répertoire des substrats endogènes de SUMO dans des macrophages stimulés ou non avec des pathogènes. Au total, 1232 substrats ont été identifiés, dont 30 différentiellement SUMOylés. La validation et la caractérisation fonctionnelle de ces substrats donnent un aperçu du mécanisme par lequel SUMO régule l'immunité. De plus, il est connu que SUMO agit au niveau de la chromatine et fonctionne comme un régulateur de l'identité cellulaire. Une autre partie de mon projet vise donc à évaluer le rôle de la SUMOylation dans un processus physiopathologique associé aux changements de destin cellulaire. Plus précisément, nous avons étudié le rôle de SUMO dans le système musculaire squelettique et montré qu’une hypoSUMOylation améliore la régénération musculaire. Nous espérons que ces études éclaireront les fonctions de SUMO dans la régénération tissulaire et permettront l'identification de nouveaux traitements contre le cancer
Immunotherapy has recently emerged as a promising approach for cancer treatment but it only benefits to a limited number of patients. Since loss of SUMOylation in immune cells induces a massive type I interferon (IFN-I) response and IFN-I-based innate responses were found to synergize with checkpoint blockade for the rejection of tumors, we investigated the effect of manipulating the SUMO pathway on anti-tumor immune responses. My results revealed that hypoSUMOylated mice show a significant delay in tumor growth upon PD-1 blockade as compared to the WT and that this anti-tumor response partly depends on IFN-I signaling and involved both myeloid and lymphocytic populations. We then identify the repertoire of endogenous SUMO substrates in macrophages either unstimulated or upon pathogenic stimuli. In total, 1232 substrates of SUMO were identified, 30 of which were differentially SUMOylated upon immune stimuli. Validation and functional characterization of these substrates give some insight into the mechanism by which SUMO regulates immunity. Moreover, it is known that SUMO acts at the chromatin and functions as a general safeguard of cell identity. In this context, another part of my project seeks to assess the function of SUMOylation in a patho-physiological process associated with cell fate changes. More precisely, we studied the role of SUMO in the skeletal muscle system and have shown that hypoSUMOylation improve muscle regeneration. We anticipate these studies to provide new insight into SUMO functions involved in the tissue regeneration as well as to allow the identification of new potential strategies to manipulate inflammation for cancer therapeutic purposes

USP28 és un enzim de-ubiquitinasa (DUBs), homologa a USP25, que pertany a la família USPs. USP28 s’ha involucrat en apoptosis induïda per IR i en l’estabilitat de vari regulador de DDR (Reparació de Dany al DNA). D’altra banda, SENP5 és una proteasa especifica per Sentrin/SUMO de la família de SENP, que s’ha descrit que està involucrada en mitosi i citocinesi. En aquesta tesi, el principal objectiu ha estat la resolució de les seves estructures cristal·lines i la caracterització de la regulació de la seva activitat proteolítica. Respecte a la part de USP28, hem aconseguit produir diferent constructes del seu Domini Catalític (CD) i de la regió N-terminal (NT), i hem caracteritzat les seves activitats sobre diferent substrats poli-Ubiquitina. També hem identificat el lloc de SUMOilització primari de USP28 per Espectrometria de Masses (K99). També hem aconseguit obtenir cristalls del NT de USP28 i del CD de USP25. Desafortunadament, la difracció d’aquests cristalls és dèbil i la seva estructura no s’ha pogut resoldre. També hem identificat l’especificitat de cadena di-Ubiquitin en USP28 (K11, K48 i K63 cadenes). Els nostres assajos in vitro indiquen que l’activitat proteolítica de USP28 es pot regular per modificació covalent per SUMO a la regió N-terminal. De totes formes, la presencia de la regió N-terminal no és estrictament necessària per l’activitat USP28. Si considerem que tenim un domini SIM, un domini UBA i dos dominis UIMs a la regió N-terminal, fóra interessant estudiar aquest aspecte amb més detall en el futur. Respecte a la part de SENP, hem posat molts esforços en la producció d’una construcció soluble del Domini Catalític (CD) de SENP3 i SENP5. Finalment, hem aconseguit produir en grans quantitats un constructe soluble de SENP5-CD en E.coli. Hem caracteritzat in vitro les reaccions de processament de SUMO i de deconjugació de SUMO. També ham format complexes entre un mutant inactiu de SENP5-CD-C712S amb el precursor de SUMO2 (Sp5-S2p) i amb el substrat RanGAP1-SUMO2. Només hem aconseguit obtenir cristalls de Sp5-S2p, malauradament la difracció era tan dèbil que no hem pogut resoldre l’estructura a alta resolució.
USP28 is a member of a family of deubiquitinating enzymes (DUBs), homologous to USP25, belonging to the USP family. USP28 has been involved in IR-induced apoptosis and in the stability of numerous DDR regulators. On the other hand, SENP5 is a member of the Sentrin/SUMO-specific proteases (SENP) in humans, which is reported to be involved in mitosis and/or cytokinesis. In this thesis, the main goal has been to elucidate their crystal structures and to characterize the regulation of their proteolytic activities. With respect to the USP28, we have produced different constructs of their Catalytic Domain (CD) and N-terminal región (NT), and have characterized their activities against different poly-Ubiquitin substrates. We have also identified the USP28 SUMOylation primary site by Mass Spectrometry (K99). We have been able to obtain nice crystals of the NT of USP28 and of the CD of USP25. Unfortunately, the diffractions of these crystals were weak and the structure could not be solved. We have also identified the di-Ubiquitin chain specificity of USP28 (K11, K48 and K63 linkages). Our in vitro analysis indicates that the USP28 proteolytic activity can be regulated by covalent SUMO modification at the N-terminal region; however, the presence of this N-terminal region is not strictly necessary for the USP28 activity. Considering there are one SIM, one UBA and two UIMs in the N-terminal region, this issue would be interesting to be explored deeply in the future. Regarding to the SENP part, we have put many efforts on the protein expression of different constructs of the Catalytic Domains (CD) of SENP3 and SENP5. Finally we have been able to produce in high yields a soluble construct of SENP5-CD in E.coli. We have characterized the SUMO processing and SUMO deconjugation reactions of SENP5-CD. We also have formed complexes between the inactive mutant of the SENP5-CD-C712S with SUMO2 precursor (Sp5-S2p) and with RanGAP1-SUMO2 substrates. We could only get crystals of Sp5-S2p, however the diffraction was so weak that we could not solve the structure at high resolution.

Organisms with renewable tissues require mechanisms to prevent the development of cancer. One such mechanism is cellular senescence, which irreversibly arrests the growth of cells at risk for neoplastic transformation. In this study, we show that 100 μM 5-bromo-2-deoxyuridine, 0.5 μM camptothecin, 0.5 μM aphidicolin and 2.5 mM thymidine cause chemically-induced premature senescence in different human cancer cell lines and they induce an increasing conjugation of SUMO-2/3 isoforms. Chemically- induced premature senescence also induces formation of SUMO-1 and SUMO-2/3 foci, which are colocalized with PML nuclear bodies. In addition, we describe that aphidicolin induces premature senescence in stable HeLa cell line expressing His6-tagged SUMO-1. HeLa-His6-SUMO-1 cell line has decreased number of PML bodies, which do not colocalize with SUMO-2/3 foci. Moreover, the number of PML bodies in HeLa cell line with ectopic expression of His6-SUMO-1 is not increasing during aphidicolin-induced senescence. This demonstrates that increasing number of PML nuclear bodies is not essential for aphidicolin-induced senescence. Absence of SUMO-2/3 foci and increased number of PML nuclear bodies support the theory that SUMO-1 acts as a SUMO-2/3 polymeric chain terminator. On the other hand, in stable HeLa cell lines expressing...

The aim of this diploma thesis was to study the role of posttranslational modifications of phosducin and their role in the interaction with the 14-3-3 protein as well as the influence of the complex formation on these modifications. Phosducin is a 33kDa protein commonly present in photoreceptor cells of the retina as well as other tissues. Despite many experiments, its physiological functions are still elusive. It has been speculated that fosducin plays an important regulatory role in visual phototransduction pathway, regulation of blood pressure and expression of G-proteins. The phosducin function is regulated through binding to the 14-3-3 protein, a regulatory protein involved in many biochemical processes. Phosducins binding to 14-3-3 protein requires phosphorylation of two serine residues Ser-54 and Ser-73 within the N-terminal domain of phosducin. However, the role of the 14-3-3 protein binding in the regulation of phosducin function is still unclear. In this diploma thesis proteins 14-3-3ζ∆C and phosducin (mutation Q52K) were successfully expressed and purified. The effect of the complex formation on phosducin posttranslational modifications was investigated using limited proteolysis and dephosphorylation. These experiments revealed that the complex formation significantly slowed down both...